The acidic lumenal pH in the epididymis and vas deferens is largely generated by a bafilomycin-sensitive H-ATPase on the plasma membrane and intracellular vesicles of a population of specialized epithelial cells that resemble kidney collecting duct intercalated cells in many respects. This acidic pH plays a critical role in sperm maturation and helps maintain sperm in a quiescent state until ejaculation: defective acidification may impair reproductive function in pathologic conditions, while intervention to manipulate acidification male be used to control male fertility. The aim of this proposal is to develop a thorough understanding of the regulatory mechanisms involved in this acidification process. The central hypothesis behind the proposed studies is that the differentiation and functional activity of these proton pumping cells is regulated physiologically, that vesicle recycling is involved in controlling the plasma membrane content of H-ATPase, and that transepithelia proton secretion involves parallel ion transporting proteins. A unique aspect of the proposed studies is the use of a multidisciplinary approach that will benefit greatly from the combined expertise of participants in the other projects of this PPG application. Cell and molecular biology, and immunocytochemistry will be combined with measurements of transepithelial ion flux using ion- specific vibrating probe technology, and single-cell spectrofluorimetric analysis, to identity active transporters in the intact epithelium. A wide variety of antibody and cDNA reagents are available for these studies, and a carbonic anhydrase II-deficient mouse model with potentially defective luminal acidification will also be examined. Specific aims are: 1) To define the pathways of proton secretion in normal epididymis and vas deferens, and the parallel transport proteins (e.g., Na/H and anion exchangers; Na-HC03 and NaK/2C1 co-transporters) that are involved: 2) To define the cell biological and physiological regulatory mechanism(s) of H-ATPase recycling in the epididymis and vas deferens, including the role of the cytoskeleton, GTP-binding proteins, acid-base balance and hormones: 3) To follow the development of PP-rich cells and acidification capacity in excurrent ducts during postnatal maturation. Normal animals and carbonic- anhydrase-deficient mice will be used for these studies. These studies will provide important information on the poorly understood process of reproductive tract acidification, as well as exploiting a novel epithelial model to dissect and distinct intracellular vesicle recycling pathway.